The double-stranded (ds) RNA-activated protein kinase, PKR, is one of several proteins induced by interferon and plays a pivotal role in the cellular antiviral response. PKR has also been implicated in other cellular processes including transformation, differentiation and apoptosis. There are also structural and functional connections between PKR and the RNA interference (RNAi) pathway. PKR is synthesized in a latent state and is activated upon binding to dsRNA to undergo autophosphorylation reactions that activate the kinase. In turn, activated PKR phosphorylates eukaryotic initiation factor 2a, resulting in the inhibition of protein synthesis in virally-infected cells. The importance of this antiviral pathway is highlighted by the diverse mechanisms that viruses have evolved to combat PKR. The broad objective of our research program is to define the molecular mechanisms for activation and inhibition of PKR. We will define the stoichiometries, affinities and free-energy coupling that govern formation of macromolecular complexes that regulate PKR activity using quantitative biophysical and structural methods. We will determine how NS5A from hepatitis C virus and NS1 from influenza virus interact with PKR to evade the antiviral pathway. Mutations in these proteins that affect virulence or confer interferon resistance will be correlated with PKR binding and inhibition. We will determine how short, heparin oligosaccharides function as PKR activators. Microarrays of synthetic oligosaccharides will be screened and structure-activity relationships will be generated to define novel small-molecule activators of PKR. These studies will provide the foundation for the design of therapeutic agents that target PKR for the treatment of viral infections and cancer.